Protein kinases are allosteric enzymes involved in cell signaling pathways and many pathological diseases (cancer, diabetes, rheumatoid arthritis, cardiomyopathy, and others). In the first grant period, we laid out the basis for understanding allostery in the catalytic subunit of protein kinase A (PKA-C), a ubiquitous kinase that regulates a plethora of vital cellular functions. Using a combination of NMR, X-ray and an array of other bio- physical techniques, we described the intra-molecular allosteric communication of PKA-C, showing how distal mutations affect substrate recognition and product release. Importantly, we defined the structural and dynamic signatures for activated and inhibited states of the kinase. In a recent breakthrough in our laboratory, we were able to reconstitute the full holoenzyme for NMR studies. We are now poised to study regulation and inhibition of PKA-C at the atomic level. In this competitive renewal, we propose to study the allosteric mechanism of regulation of PKA and its dysfunctional states linked to somatic mutations. Specifically, Davide Calebiro at the University of Wrzburgh (Germany) discovered a series of PKA-C mutations correlated with adenoma-associated Cushing's syndrome (AA-CS). Also, Sandy Simon's laboratory at The Rockefeller University discovered a chimera of PKA-C that is implicated in the progression of the fibrolamellar hepatocellular carcinoma (FLHCC). To study the effects of these mutations, we formed a consortium with Susan Taylor (UCSD), and will collaborate with the Calebiro and Simon groups to elucidate the changes in the intra- and inter-molecular signaling generated by these disease mutations using a combination of NMR spectroscopy, thermodynamics and kinetic methods. In AIM1, we will probe the role of PKA-C in the assembly of the holoenzyme and how the AA-CS mutants affect the R/C complex. In AIM2, we will analyze how AA-CS mutations affect the regulation of PKA-C by endogenous and exogenous inhibitors. In AIM3, we will study the structure and the dynamics of the chimeric construct of PKA-C (PKA-CDNAJB1) to understand how the fused chaperone affects pseudo-substrate recognition and regulation, degenerating into FLHCC. Since the ground states of these mutants are identical, the different functions must be encoded in the excited states that only NMR spectroscopy can unveil at the atomic resolution. Understanding the changes in the kinase energy landscape caused by these mutations is key to the elucidation of the molecular mechanisms leading to disease. This information will help the design of new strategies to control kinase activity for innovative therapies.